Electrospray ionization source and method for mass spectrometric analysis

An electrospray ionization probe assembly for introducing a sample of ions into a mass spectrometer for mass spectrometric analysis, said sample being generated by an electrospray source generating a spray of charged droplets containing molecules of interest and solvent, said probe assembly comprising:

• a desolvation tube having an entrance orifice and an exit orifice;
• means for applying a first voltage to said desolvation tube;
• means for heating said desolvation tube;
• a thermocouple for measuring the temperature adjacent the exit orifice of said desolvation tube;
• a skimmer for focusing and directing said ions to the mass spectrometer, said skimmer having a second voltage applied thereto and an orifice electrically isolated from the desolvation tube, said skimmer orifice being positioned at a fixed distance from the exit orifice of said desolvation tube;
• an electrically conductive lens positioned in front of said skimmer for initially focusing said ions after their exiting from the desolvation tube and prior to their entering the skimmer orifice, said lens having an adjustable engagement with said desolvation tube such that the distance between the lens and the skimmer orifice is variable;
• and
• an evacuable dielectric housing for housing said desolvation tube, heating means, thermocouple, skimmer, and lens.

A system for analyzing the mass spectra of molecules of interest, comprising:

• a mass spectrometer having a lens stack and an inlet orifice for receiving therethrough ionized molecules of interest to be analyzed;
• and
• an electrospray ion source adapted to be sealingly received within the inlet orifice of said mass spectrometer for introducing ionized molecules of interest therein for analysis, said electrospray ion source including:
• a source of a dilute solution of the molecules of interest;
• electrospray means for spraying tiny charged droplets of said solution;
• means for imposing a first voltage on said electrospray means;
• a desolvation tube having an entrance orifice positioned across a gap from said electrospray means for receiving said charged droplets and an exit orifice for ionized molecules of interest;
• means for imposing a second voltage on said desolvation tube;
• means for heating said desolvation tube;
• thermocouple means for monitoring the temperature of said desolvation tube;
• a sampling cone having a variable voltage applied thereto for directing said ionized molecules of interest to the mass spectrometer, said sampling cone having an outlet orifice and an inlet orifice, said inlet orifice being electrically isolated and positioned at a first distance from the exit orifice of said desolvation tube;
• a lens positioned for initially focusing said ionized molecules of interest after exiting the desolvation tube and prior to entering said sampling cone, said lens being adjustably affixed to the desolvation tube adjacent the exit orifice thereof such that the distance between said lens and the inlet orifice of said sampling cone is variable;
• an evacuable tubular encasing for housing said desolvation tube, heating means, thermocouple means, sampling cone, and lens, said mass spectrometer having a vacuum chamber in communication with the inlet orifice of said mass spectrometer, said inlet orifice of said mass spectrometer forming a vacuum seal with said evacuable encasing adjacent the outlet orifice of the sampling cone;
• and
• means for creating a vacuum in said mass spectrometer, inlet orifice and evacuable encasing.

A method for introducing desolvated or partially desolvated ionized molecules of interest into a mass spectrometer for analysis, said method comprising:

• creating a dilute solution of molecules of interest in a solvent;
• charging said solvent and molecules of interest;
• generating a fine spray of tiny droplets of said dilute solution of molecules of interest and solvent;
• providing a desolvation tube having an entrance orifice and an exit orifice;
• providing a sampling cone downstream of the exit orifice of said desolvation tube;
• positioning the entrance orifice of said desolvation tube adjacent the point of generation of the fine spray of tiny droplets;
• applying a first voltage to said desolvation tube;
• applying a second voltage to said sampling cone, said second voltage being equal to or lesser than the first voltage applied to said desolvation tube;
• receiving said charged droplets of said dilute solution in the entrance orifice of said desolvation tube;
• transporting said droplets to the exit orifice of said desolvation tube;
• controllably heating said desolvation tube to partially or substantially desolvate said droplets during their transport therethrough to provide ionized molecules of interest at the exit orifice of said desolvation tube;
• focusing said ionized molecules of interest utilizing a lens positioned upstream from said sampling cone, said lens having a central orifice extending therethrough disposed in axial alignment with the exit orifice of said desolvation tube, said lens comprising an electrically conductive element having an adjustable engagement with said desolvation tube such that a distance between said lens and said sampling cone is variable;
• and
• directing the focused ionized molecules of interest upon their exit from the lens through said sampling cone, said sampling cone having an orifice extending therethrough disposed in axial alignment with the exit orifice of the desolvation tube and the central orifice of said lens, whereby the voltage differential between said desolvation tube and said sampling cone electrostatically focuses and selects ions of proper kinetic energy for transport to the mass spectrometer.

A method for characterizing the three-dimensional structure of a protein molecule, said method comprising:

• (a) performing electrospray ionization mass spectrometry (ES-MS) to obtain the spectrum of a protein-small molecule complex, said ES-MS being performed as follows:
• creating a solution of a small molecule, a larger protein molecule, and protein/small molecule complexes;
• charging said solution and molecules of interest;
• generating a fine spray of tiny droplets of said solution;
• providing a desolvation tube having an entrance orifice and an exit orifice;
• positioning the entrance orifice of said desolvation tube adjacent the point of generation of the fine spray of tiny droplets;
• applying a voltage to said desolvation tube;
• receiving said charged droplets in the entrance orifice of said desolvation tube;
• transporting said droplets to the exit orifice of said desolvation tube;
• controllably heating said desolvation tube to substantially desolvate said droplets during their transport therethrough to provide ionized protein molecules at the exit orifice of said desolvation tube;
• focusing said ionized protein molecules after exiting the desolvation tube between said exit orifice and a mass spectrometer utilizing a lens positioned adjacent the exit orifice of said desolvation tube, said lens having a bore extending therethrough in axial alignment with the exit orifice of said desolvation tube;
• directing the focused ionized protein molecules upon their exit from the lens through a skimmer to remove inadequately ionized protein molecules, said skimmer having an orifice extending therethrough in axial alignment with said exit orifice of said desolvation tube and the axial bore of said lens;
• and
• analyzing the ionized protein molecules in a mass spectrometer to obtain said spectrum;
• (b) using said spectrum from step (a) to calculate the binding constant K.sub.B for the binding of the small molecule to the protein;
• (c) repeating steps (a) and (b) with additional different small molecules;
• (d) calculating the heat of formation .notident.H.sub.f for the binding of each of the small molecules used in steps (a)-(c) to a selected residue on the protein;
• (e) repeating step (d) for other selected residues on the protein;
• (f) comparing the K.sub.B values calculated in steps (b) and (c) with the .DELTA.H.sub.f values calculated in steps (d) and (e);
• and
• (g) utilizing the comparisons of step (f) to characterize the three-dimensional structure of the protein.